Bindslev-Jensen C

Position Paper

European Academy for
Allergology and Clinical Immunology

First Draft June 2000

Introduction

Introduction of genetically
modified foods to the consumers carry a potential risk of introducing allergic
reactions in susceptible individuals with food allergy (1). Although the risk
and significance of the much quoted case, where allergenic proteins from Brazil
Nut was introduced in soy for chicken feed (2) has been overestimated, new
genetically modified plants with a real allergenic risk are now being
developed. As an example,the allergen β-casein from cows milk has been
inserted into soy bean and glycinins from soy into rice (3,4).

Although these
products are not (and probably never will reach) the market, the risk of
contamination or spreading of genetic material always exist.

Therefore, the
necessity of control procedures for ascertaining potential allergenicity in
GMO-foods is obvious. Legislation in Europe (and in US) demands such proce­dures,
but no mutual agreement upon the practical aspects has been reached so far.

A Decision Tree has
been launched by FCBS/ILSI (5), aiming at enabling an evaluation for labelling
of a GMO-foods based on available data on the insert protein, but a flow chart
for risk evaluation does not exist at present.

This position Paper
aims at reviewing the present knowledge within the field of GMOs and
allergenicity and at presenting a suggestion for ascertainment of allerge­nicity
in genetically modified foods.

The following is an
attempt to give an overview of the EC regulation concerning the safety
assessment and labelling of genetically modified organisms (GMOs) with special
emphasis on allergenicity. The description is restricted to EC regulation not
because it is not recognised that there are countries in Europe not members of
the EU but because the Eu constitutes the largest group of countries in Europe
and is likely to expand in the future.

Novel foods

Rules concerning GMOs
are covered in the regulation dealing with novel foods (NF) (1). Novel foods
are defined as foods and food ingredients, which have not hitherto been used
for human consumption to a significant degree within the Community and which
fall under the following categories:

(b)foods
and food ingredients produced from, but not containing, genetical­ly modified
organisms;

(c)foods
and food ingredients with a new or intentionally modified primary molecular
structure;

(d)foods
and food ingredients consisting of or isolated from micro-orga­nisms, fungi or
algae;

(e)foods
and food ingredients consisting of or isolated from plants and food ingredients
isolated from animals, except for foods and food ingredients obtained by
traditional propagating or breeding practices and having a history of safe use;

(f)foods
and food ingredients to which has been applied a production process not
currently used, where that process gives rise to significant changes in the
composition or structure of the foods or food ingredients which affect their
nutritional value, metabolism or level of undesirable substances.

From the above it will
be clear that the regulation is designed to cover a variety of different
situations and that GMOs are only a subset of novel foods.

It is stated that
foods and food ingredients falling within the scope of the regulation must not:

-present a danger for the consumer,

-mislead the consumer,

-differ from foods or food ingredients which they are intended to
replace to such an extent that their normal consumption would be nutritionally
disadvantageous for the consumer.

Before marketing a
novel food

The manufacturer
wanting to place a GMO on the Community market shall submit a request to the
Member State in which the product is to be placed on the market for the first
time. At the same time, he shall forward a copy of the request to the Com­mission.
The request shall contain the necessary information, including a copy of the
studies carried out and any other material which is available to demonstrate
that the food or food ingredient do not present a danger to the consumer,
mislead the consumer, etc. The request shall be accompanied by a summary of the
dossier. This summary shall be sent to the other Member States without delay.

The Member State
concerned shall notify the Commission of the competent food assessment body
responsible for preparing the initial assessment report. This report must be finished
within three months from receipt of a request. The report shall be send to the
other Member States. After that and within 60 days a Member State or the
Commission may make comments or present a reasoned objection to the marketing
of the food or food ingredient concerned.

Notification procedure

Food or food products
that are substantially equivalent to existing foods do not have to go through
the above procedure. Here it is just necessary to notify the Commis­sion
together with the relevant details, including the confirmed statement that the
products are, in fact, substantially equivalent. This statement should be made
by an official food assessment body in one of the member states. Products of
this nature may be marketed straight away, i.e. immediately after being
notified to the Commis­sion. A GMO can not be substantially equivalent, but a product madefrom a GMO can, e.g. soybean oil and maize flour or starch.

Information necessary
for safety assessment

The EU Scientific Committee
for Food (SCF) has developed recommendations concerning the scientific aspects
of the information necessary to support an applica­tion, how such information
must be presented and how the initial report must be prepared (2).

SCF has suggested structured
schemes to identify the types of information that are likely to be required to
establish the safety of particular classes of novel foods. It is underlined
that the schemes only can be used for guidance. It must be decided on a case by
case basis what precise information is needed.

The information
requested is:

I0Specification of the novel food (NF)

II0Effect of the production process applied to
the NF

III0History of the organism used as the source of
the NF

IV0Effects of the genetic modification on the
properties of the host organism including,

-characterisation of the parent food organism,

-characterisation at the molecular level of
the nature of the genetic modification includinginsertional position, copy number and
biochemical expression level,

-establishment, as far as possible, of
substantial equivalence between the parent food organism and its new derivative
through chemical and phenotypic analysis,

The concept of substantial equivalence embodies the
idea that existing organisms used as foods can serve as basis for comparison
when assessing the safety of human consumption of a food that has been modified
or is new. If a new food is found to be substantially equivalent to an existing
food, it can be treated in the same manner with respect to safety, keeping in
mind that establishment of substantial equivalence is not a safety or
nutritional assessment in itself, but an approach to compare a potential new
food with its conventional counterpart.

-if substantial equivalence cannot be established, conventional safety
studies on specific chemicals occurring in the food due to the phenotypic
change involving either the new product of the new gene or the safety of
inherent natural toxins now present in altered amounts. The potential
allergenicity of the new components also needs to be addressed.

IX.Genetic
stability of the GMO used as NF source

X.Specificity
of expression of novel genetic material

XI.Transfer
of genetic material from GMO

XII.Ability
of GMMicroorganisms to survive in and colonise the human gut

XIII.Anticipated
intake/extent of use of the NF

XIV.Information
on previous human exposure to the NF or its source

XV.Nutritional
information on the NF

XVI.Microbiological
information on the NF

XVII.Toxicological
information on the NF

If substantial
equivalence to a traditional counterpart cannot be established the safety
assessment based on a case-by-case evaluation must consider the following
elements:

-consideration of the possible toxicity of the analytically
identified individual chemical components,

-toxicity studies in vitro
and in vivo including mutagenicity
studies, reproduction and teratogenicity studies as well as long term feeding
studies, following a tiered approach on a case-by-case basis,

-studies on potential allergenicity.

The allergenicity of a
novel food from a GM source should include consideration of the allergenic
potential of the donor and of the recipient organism. It is suggested to do in vitro and in vivo tests in individuals allergic to the traditional food
counterpart, although it is recognised that it raises ethical problems. If the
novel protein comes from a source that is known to be a food allergen specific
immunological test with sera from allergic individuals are suggested. If these
tests are negative, in vivo skin prick
tests and oral challenges may be performed. It is recommended to include
factors such as sequence epitope homology with known allergens, heat stability,
sensitivity to pH, digestibility by gastrointestinal proteases, detectable
amounts in plasma, and molecular weight as possible indicators of
allergenicity. Additional evidence might emerge from pre-marketing human
results and reports of workers sensitisations. As with the other toxicological
endpoints decision on which test it is possible and necessary to perform should
be done on a case-by-case basis. It is stated that new approaches are needed to
assess the potential allergenicity of NF in humans.

References to this
chapter:

1 Regulation (EC) No
258/97 of the European Parliament and of the Council of 27 January 1997
concerning novel foods and novel food ingredients.

2 Commission
recommendation of July 1997 concerning the scientific aspects and the
presentation of information necessary to support applications for the placing
on the market of novel foods and novel food ingredients and the preparation of
initial assessment reports under Regulation (EC) No 258/97 of the European
Parliament and of the Council.

Methods used in
the production of transgenic plants

Until roughly 15 years
ago, conventional plant breeding was the only way to improve agricultural
productivity and nutritional quality of food crops. By intercrossing varieties
with different desired characteristics new improved varieties were (and are)
created. The introduction of molecular biology in the field of plant research
has resulted in the possibility to introduce selected genes from almost any
life form into plants by genetic engineering. This development has increased
the diversity of genes available for introduction and decreased the time needed
for the development of new varieties. At the same time, complete genomes of
living organisms are becoming available. The number of candidate genes for
introduction in plants will, therefore, increase exponentially. Moreover, the
elucidation of plant genomes will supply us with valuable new information on
the regulation of expression of proteins in plants, facilitating new strategies
of regulated protein expression. How are new genes introduced into plants? In
principle there are two classes of plant transformation technologies: natural
and non-natural QUOTE{ ADDIN REFMAN
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. The natural methodologies use natural pathogens of plants for
introducing foreign genes, i.e. viruses and bacteria. The non-natural
approaches are based on physico-mechanical techniques.

Plant transformation
technologies

Agrobacterium tumefaciens-mediated gene transfer

Agrobacterium tumefaciens is a natural pathogen for
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1;2 QUOTE{ ADDIN
REFMAN
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. It invades wounds in plant tissue, where it introduces small segments
of its own DNA, the so-called Ti-DNA (tumor-inducing DNA). This results in
local production of phytohormones that induce tumor growth, the so-called crown
galls that function as sites of refuge for the bacterium. For the introduction
of heterologous genes in plants, A.
tumefaciens was made non-pathogenic by removing genes involved in tumor
induction. The present A. tumefaciens
plant transformation vectors are also suitable for replication in E. coli, allowing convenient
manipulations. Usually the vector contains two resistance genes, one for
selection in bacteria (e.g. spectinomy­cin) and one for selection in plants
(e.g. kanamycin or a herbicide). The gene of interest is inserted behind a
plant promotor sequence like the cauliflower mosaic virus 35S promotor (CaMV 35S).A.
tumefaciens can be used for transforming plant cells as well as plants.
After transfer, the part of the DNA is integrated into the genome of the plant
and the desired protein is expressed. The fragment that is inserted, T-DNA
(transferred DNA), is flanked specific regions called border sequen­ces.
Recognition of these sequences is essential for injection of DNA into the plant
cell. The expression of heterologous genes has been shown to be stable for at
least 5 generations. The integration into the plant genome can occasionally
also be a disadvantage, because plant genes could be disturbed. Finally, most
monocotelydo­nous plants are no natural host for A. tumefaciens. This implies that this method for plant
transformation is not suitable for a number of very important food crops, i.e.
cereals like wheat, rice and corn.

Physico-mechanical
transformation methods

For transformation of
monocotelydonous plants, originally protoplasts (plant cell from which the cell
wall has been removed) were used QUOTE{
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. Several techniques have been applied to protoplasts including
electroporation, microinjection, polyethylene glycol treatment, and calcium
phosphate treatment. Unfortunately, it is hard to regenerate plants from
protoplasts. This can, however, efficiently be achieved from immature embryos.
To transform embryonic cells from cereals the particle gun method or
high-velocity microprojectile bombardment was introduced QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\031‑3\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\09\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03496\03496\00\03\00
1-3 QUOTE{ ADDIN
REFMAN
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. This is now the most common technique used for cereals. Microscopic
gold or tungsten particles coated with DNA are accelerated by explosive charge,
high pressure helium, or by electric discharge, towards the embryonic cells.
This enables the particles to pass the rigid cell wall and enter the cytoplasm.
In this method, the transformed genes are also integrated into the plant
genome, resulting in very stable transformation. As with A. tumefaciens selection of transformed cells is achieved by
co-transformation of resistance genes.

In order to alter
agronomic characteristics of food crops it will be necessary to manipulate
complex metabolic pathways that are regulated by multiple genes. This requires
the integration of multiple transgenes into the plant genome. Although possible
with A. tumefaciens, it is very
complex and demanding. Microprojectile bombardment has now been show to be an
efficient method to introduce multiple genes. By mixing different genes and a
sinlge selectable marker, up to 13 genes were simultaneously transformed to
rice by so called cobombardment QUOTE{
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4.

Antisense approach:
towards hypo-allergenic foods?

The methods described
so far aim at introducing new genes into plants. Sometimes it may be desirable
to suppress a gene. A method that has been successfully used in achieving this,
is the antisense technique QUOTE{ ADDIN
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2. The method is based on the fact that DNA transcription is
a unidirectional process, from the 5 to the 3 end along the so-called sense
strand. By transforming plant cells with an inverted gene, the transcri­bed
sequence will be identical to the antisense strand. The cell will contain both
the normal gene and an inverted version. As a result, both sense and antisense
mRNA will be produced. These molecules will hybridize because their sequences
are exactly complementary. Translation of mRNA is prevented by the interference
of the antisense mRNA. This technique has for example been used to slow down
the ripening of fruits like tomato and melon by knocking out the gene for ACC
oxidase QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\035;6\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00\01\00\00\01\0D\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03500\03500\00\03\00
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. This enzyme catalyzes the last step of ethylene biosynthesis. Ethylene
is thought to trigger several processes observed during ripening. The antisense
approach might in the future prove useful to suppress genes for allergens.

Present
situation of the use of genetically modified foods in the food chain

Microbial Products and
Microorganisms

Independent of genetic
engineering, microorganisms are used for biotechnological production of food
additives for many years. Examples for food additives produced by bacteria,
yeasts or moulds are amino acids, flavourings , organic acids, hydrocol­loids,
preservatives, and vitamins [1-3]. Current approaches of genetic engineering
are targeted towards (i) increased production (ii) replacement of chemical
synthesis by cheaper fermentation processes (iii) modifying the characteristics
of microorga­nisms and (iv) design of new products such as enzymes with improved
properties. Most of these products are still under development. However, many
enzymes derived from genetically modified microorganisms are being used by the
food industry (Table 1). These enzymes do not differ from their natural
homologues as regards structure and function. Bacteria, yeasts and moulds that
are recognised as safe are used as host organisms [2,4]. Compared to
conventional production of these enzymes, use of GMO-derived products saves
energy and other resources.

Moreover,
microorganisms are widely used as starter cultures for fermented foods and
beverages. In 1990, a genetically modified yeast was approved in the UK [5].
This yeast presented enhanced maltase and maltose permease activity, resulting
in improved fermentation of maltose and improved dough-leavening
characteristics.However, this organism
is not used on a commercial scale.

In 1994, another
genetically modified yeast obtained approval from the UK authoriti­es for use
in the production of pasteurised beer [6]. The yeast contains a glucoamy­lase
gene from a different yeast species, resulting in increased degradation of
dextrins, reduced carbohydrate, and increased alcohol content. By
pasteurisation, viable yeast cells are deactivated in the final product.

Plants and Plant
Products

Genetically modified
herbicide tolerant soybeans developed by Monsanto obtained approval for use in
food products within the European Union in April 1996. This decision was based
on directive 90/220/E­EC [7] which is a part of the general legislation
regulation the application of gene technology. Insect-resistant maize developed
by Ciba-Geigy (now Novartis) was released on the basis of the same directive in
January 1997. Since then, food products containing these transgenic foods can
be marketed in the EU. Moreover, in 1996, national approval was obtained in the
UK for tomato puree produced from delayed ripening tomatoes and products from
transgenic maize.

In May 1997 a European
Community Regulation was established applying to novel foods including those
derived from genetically modified organisms [8]. Foods and food ingredients
containing or consisting of GMO undergo an authorisation proce­dure. By
contrast, notification is required for those foods and food ingredients that
are substantially equivalent to existing foods as regards their composition,
nutritional value, metabolism, intended use and the level of undesirable
substances contained therein. According to this regulation rapeseed oil derived
from transgenic crops and additional foods and food ingredients produced from
transgenic maize have been notified and can thus be marketed within the EU
(Table 2).

In addition, the foods
listed in Table 3 (tomatoes, radicchio, chicoree, and again soybeans and maize)
have been submitted to the authorisation procedure.

Outside the EU,
genetically modified food plants have been approved in USA, Canada, Mexico,
Argentina, China, Japan, South Africa, Australia, Ukraine, and Romania [4] [9].
In 1999, transgenic crops were grown world-wide on an area of 39.9 million ha,
with 74 % in the USA, 6.7 % in Argentina, and 4.0 % in Canada. 71 % of the
plants were herbicide-tolerant, 22 % insect-tolerant, 7 % were resistant to
both herbicides and insects, and less than 1 % were virus-resistant [9].

Table 2: Authorisations/notifications for the placing on the
market of foods derived from genetically modified organisms.

Table 3: Applications for the placing on the market of
genetically modified organisms and derived foods according to Regulation (EC)
No. 258/97.

Applicant

Description of

Food or Food Ingredient

Application in

Authorisation

Zeneca

Genetically
modified processing to­matoes

ES

pending

Bejo
Zaden

Transgenic Radicchio rosso with ma­le sterility

NL

pending

Bejo
Zaden

Transgenic
green hearted chicoree with male sterility

NL

pending

DuPont

High
oleic soybeans

NL

pending

Monsanto

Herbizide
tolerant maize GA21

NL

pending

Plant
Genetic Sy­stems

Herbizide
tolerant soybeans

BE

pending

Novartis

Insect
tolerant sweet maize Bt 11

NL

pending

In addition to the
plants and foods approved by the EU members, e.g corn, rapeseed and soybean
crops with resistance to other insects and herbicides have been released.
Further examples are various tomatoes with delayed softening, delayed ripening,
or virus resistance, insect resistant potatoes, and virus resistant squash and
papaya. The first product with modified physiological properties was a high
laureate rapeseed that was approved 1995 in the USA and 1996 in Canada [4]. In
contrast to the first generation of transgenic crops which almost exclusively
had improved agronomic properties, there is an ongoing agronomic trend towards
introducing benefits for the consumer by improving the nutritional value. High
oleic soybean with the potential to reduce the bad blood cholesterol, which
has been released in the United States is another example for such a food [9].
There is a rapid development in this area. A popular example is the
introduction of genes facilitating the synthesis of beta-carotene, the precurser
of vitamin A, into rice. Keeping in mind that Vitamin A deficiency causes the
death of 2 Million children in developing countries every year, this could be a
substantial health benefit in countries with a rice-based diet [9].

Animals

The most important efforts
to generate genetically modified animals have been made with fish [4, 10].for
herbicide-tolerant soybeans. Eleven of the produtcs were found to contain
roundup ready soybean, but only one product was labelled as containing
genetically modified material [12]. In a Norwegian study, 300 food samples were
taken. Out of 150 samples of soy-containing products, DNA could be isolated
from 117 samples. Genetically modified soybean was detected in 59 of these 117
samples (about 50 %). Six samples from ordinary grocery stores, all belonging
to one type of product, contained only traces of GMO soybean. By contrast 52
positive samples were from health food stores. 22 of these samples representing
eight different types of products contained about 2 % of GMO soy , and 24
samples with seven different categories of products contained more than 2 %
GMO-derived material [13]. Another 150 samples were taken from maize-containing
foods. DNA could be successfuly isolated from 137 of these samples. Genetically
modified maize at levels below 2 % was detected in 23 of the 137 samples (17
%); 13 of the samples were from ordinary grocery stores and 10 were from health
food stores [13].

In a German study
three out of 25 seed corn m The attempts are targeted towards e.g. improvement
of growth rates, cold tolerance, disease resistance, or optimised composition.
However, genetically modified animals have not been released to the market
either inside or outside the EU.

Declaration

GMO derived foods and food
ingredients are subject to labelling in order to inform the consumer of the
genetic modification if the recombinant DNA or resulting new proteins are
present in a proportion higher than one percent of the food or food ingredient
[11].

Detection of GMO derived foods

The analysis of
transgenic material in food samples is generally based on PCR techniques.
Analyses to detect the presence of GMO foods in commercially available food
products are underway. The first data have recently been published.

In a Swedish study 45
food samples,all labelled as containing soybean protein, soya lecithin, or
soybean oil were analysed aize samples have been shown to contain between 0.1 %
and 0.5 % insect-tolerant maize of the Monsanto variety MON 810 [14]. Moreover,
it has been shown that Canadian rapeseed honey contains pollen of
glufosinate-resistant rapeseed [15].

Conclusions

It is difficult to
estimate to which extend the released products prepared using GMO are already
present in the food chain. However the studies from Sweden and Norway have
clearly shown thatmany foods containing
soybeans and maize already contain genetically modified material [12, 13]. The
data also show that the major GMO foods, transgenic soybean and maize, are
widely used. The results obtained with Canadian rapeseed honey may indicate
that transgenic material can occur in a wider range of foods than anticipated
by the consumer.

7.
Council Directive of 23 April 1990 on the deliberate release into the
environment of genetically modified organisms (90/220/EEC), Official Journal of
the European Communities L 117, 8.5.1990, p. 15

8.
Regulation (EC) No 258/97 of the European Parliament and of the Council of 27
January 1997 concerning novel foods and novel food ingredients, Official
Journal of the European Communities L 43, 14.2.1997, p. 1

9.
James C. Transgenic foods plant: traits, transformants, and deployment. ISAA
(International Service for the Acquisition of Agri-Biotech

12.
Commission Regulation (EC) No 49/Ö2000 of 10 January 2000 amending Council
Regulation (EC) No 1139/98 concerning the compulsory indication on the
labelling of certain foodstuffs produced from genetically modified organisms of
particulars other than those provided for in Directive 79/112/EEC, Official
Journal of the European Communities No L 6/13, 11.1.2000

Molecular biology and
biochemistry have significantly increased the knowledge of the nature ofallergens. However, only limited information
about specific properties of food allergens is presently available. At present,
it is useful to classify plant food allergens according to their biological
functions into several families (Table 1). The majority of known plant food
allergens belong to seed storage proteins (1-16), protease and
amylase-inhibitors (17-22), profilins (23-27) or pathogenesis-related (PR)
proteins (28-58). Other important protein families that include plant food
allergens are the cereal peroxidases, thiol proteases and lectins (for review
see 59). Less variety is found among allergenic proteins derived from animal
sources. However, also in this case, cross-reactive allergen families can be
subdivided (60-62). These proteins will not be discussed here as they found no
use for the produc­tion of GMOs until now.

In response to
pathogens, plants synthesize and accumulate a variety of PRs which are part of
a plants defence system. As plant protection against bacteria, fungi, viruses
and insects is a major challenge to agriculture world-wide, overexpression of
PRs in transgenic plants has been applied to increase the defense potential.
Transgene-encoded, heterologous PRs are used in addition to, and not in place
of, the inducible defences of the host plant. On the other hand, genes encoding
proteins with a high content of essential amino acids, and therefore high nutritive

value, have also been
considered for the transformation of crop plants.

Proteins with
allergenic potential which are considered for use in the production of GMOs to
increase the resistance to microbial and insectal attack

Anti-fungal proteins

Transformation of crop
plants with either ß-1,3-glucanases or chitinases provides protection against
fungal attack, as both enzymes hydrolyze cell walls of several plant-pathogenic
fungi. A basic ß-1,3-glucanase found in Hevea
brasiliensis latex has been identified as Hev b 2,a relevant latex allergen (30-31). The
coincidence of Hevea latex allergy
and hypersensitivity to plant-foods, especially banana, avocado, and chestnut,
has been is called latex-fruit syndrome. Indeed, ß-1,3-glucanases from Hevea brasiliensis reacted with specific
IgE from food-allergic patients suffering from adverse reactions to banana and
other fruits (32). Chitinases are proteins present in many seed-producing
plants. In chestnut and avocado class I chitinases have been identified as
allergens (33-34). An endochitinase designated as Pers a 1, a major allergen of
avocado, was cloned and expressed in the yeast Pichia pastoris (35). It is believed that sensitization to Pers a 1
is a consequence of IgE-production against latex-hevein (36). Moreover, two
banana allergens were identified as class I chitina­ses containing a
hevein-like domain (37).

Thaumatin-like and
osmotin-like proteins possess diverse functions including antifungal activity (63).
Due to sequence similarities, the PR-group 5 proteins (28) and thaumatin (an
intensely sweet tasting protein produced by the African shrub Thaumatococcus daniellii) are now
designated "thaumatin-like proteins". Transgenic potato plants
overexpressing a thaumatin-like protein showed increased resistance to fungal
infection(63). A major allergen of
apple was the first thaumatin-like protein described as an allergen (38). The
complete cDNA sequence has been decoded and the allergen received the designation
Mal d 2 from the international nomenclature committee (Krebitz M.,
unpublished). A thaumatin-like protein representing a major allergen was cloned
from sweet cherry, and designated Pru av 2 (39). Furthermore, the N-terminal
sequence of an important bell pepper allergen was displayed a high degree of
identity with a corresponding portion of the osmotin-like protein P23 from
tomatoes (40).

Transfection of tomato
plants with prohevein (Hev b 6.01) from Hevea
brasiliensis led to a retardation of the growth of the fungus Trichoderma hamatum (64).Peptide sequences of a recently
identified turnip allergen revealed 70% identity to prohevein and high
similarities to wound-induced proteins from tomato and potato (41).

Insecticidal proteins

Besides endochitinases
(see above), trypsin-inhibitors and patatins have been considered as
transgene-candidates for the protection against insect pests. Aller­gens can be
found in all three categories.

It has been shown that
plant-derived protease-inhibitors are potent inhibitors of larval growth (65).
Concerning the use of trypsin inhibitors in transgenic plants, proof of concept
was established in non-edible plants (66). Feeding of transgenic peas
expressing a bean proteinase inhibitor to rats caused no harmful effects. The
authors concluded that such GMOs might be used in the diet of farm animals in
the future (67). The Kunitz soybean trypsin inhibitor binds IgE of soy
challenge positive patients, indicating that this protein is an allergen (17,
18). Inhibitors of proteases and alpha-amylases are also found in high
concentrations in plant storage organs such as cereal seeds or tubers (19-22).

Patatin is the major
storage protein of potato tubers and has been reported to inhibit the growth of
insect pest larvae (68). Recently, patatin has been demonstrated to represent a
major allergen of potato designated Sol t 1(9). Moreover, Hevea latex contains a patatin-like allergen with sequence homology
to Sol t 1, designated Hev b 7 (10, 69).

Resistance to bacteria

LTPs can take part in
plant defense, as some LTPs have potent antifungal and antibacterial activities
(49-50). An enhanced tolerance to bacterial pathogens was achieved in tobacco
transformed with a barley lipid transfer protein gene. The results obtained
encourage the use of this strategy also in crop plants (70)LTPs are important allergens of the
Prunoideae, such as peach, plum and cherry (51). The LTP of peach was named Pru
p 3, LTP of aplle Mal s 3, respectively, by the interna­tional allergen
nomenclature committee (52-56). IgE-mediated reactions to beer can be caused by
barley-LTP which is involved in beer foam formation (57).

Improvement of
nutritive value

Mammalian organisms
are incapable of synthesizing essential amino acids. There­fore, one of the
major targets of genetically modifying crop plants is the improvement of their
amino acid composition by introducing heterologous seed storage proteins, which
are rich in essential amino acids, such as albumins. However, several exam­ples
of albumins exist showing that these molecules also possess allergenic properti­es.
The major allergens ofSin a 1 (yellow
mustard), and Bra j 1 (oriental mustard), can elicit allergic reactions (1-3).
The 2S albumins are also found to be allergens, present abundantly in nuts,
e.g. in the Brazil nut and the English walnut (5-8). Plans for commercial
development of GMOs expressing the brazil nut 2S albumin were abandonned
because of the proteins allergenicity (71). However, the expression of an
amaranth albumin gene in transgenic potato was not associated with allergenic
complications (72).

Conclusion

The classification of
proteins into certain families according to their biological functions
facilitates the unmasking of potential allergens encoded by genes used for the
transfection of plants. Many PR proteins that represent very attractive
candidates for the production of transgenic plants are allergens. Several of
the seed storage proteins that are candiates for improving nutritive value of
crop plants have also been identified as allergens.It is suggested to apply detailed
investigations whenever proteins with these characteristics are taken into
consideration.

It is known for
approximately two decades now, that IgE antibodies of pollen allergic patients
can be directed to complex N-linked glycans on pollen glycoproteins QUOTE{ ADDIN REFMAN
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1. These IgE antibodies are extremely crossreactive between
different pollen, vegetable foods and even foods of invertebrate animal origin.
The structural basis for this high degree of crossreactivity is largely
elucidated QUOTE{ ADDIN REFMAN
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2. Essentially, plant complex N-glycans have two typical
monosaccharide substitutions that are not found in mammals: an a(1,3)-fucose
linked to the proximal N-acetylglucosamine and a b(1,2)-xylose linked to the
core mannose. These monosaccharides are pivotal in immunogenicity and allergeni­city
of plant N-glycans. There is a dispute concerning the clinical relevance of
N-glycan specific IgE antibodies. Some groups consider these IgE responses
clinically irrelevant QUOTE{ ADDIN
REFMAN
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3. Patients with carbohydrate-specific IgE antibodies
frequently demonstrate a positive in
vitro test results for vegetable foods, but they do not have clinical food
allergy. This implies that these antibody responses would preferably not be
picked up in in vitro food allergy
diagnostics QUOTE{ ADDIN REFMAN
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4. There are however also groups that have reported clear
biological activity of carbohydrate-specific IgE antibodies in basophil
histamine release assays QUOTE{ ADDIN
REFMAN #\11\05ê\19\01\00\00\00\015\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03124\03124\00\03\00
5. Whether this biological activity is linked to clinical
food allergy was left in the dark in these reports.

What is the relevance
of N-glycan reactive IgE antibodies for GMOs? If proteins with putative
N-glycosylation sites are expressed in transgenic plants, they will be
glycosyated with plant N-glycans. These structures will thus be transformed
into IgE-binding structures. This does not imply an extra allergenic risk for
GMO foods, because they are full of glycoproteins carrying IgE-binding
N-glycans anyway,with or without an additional transgene for a glycoprotein. If
the lack of biological activity of anti-carbohydrate IgE is confirmed, this has
repercussions for the selection of sera to be used for judging whether a
transgene codes for an allergen or not. Sera with anti-carbohydrate IgE will
recognize virtually any plant- orinvertebrate animal-derived glycoprotein. The candidate transgene will
then unjustly be designated as a potential allergenic risk on the basis of
recognition by anti-carbohydrate IgE. Because sera from clinically well-defined
food allergic partients are rare, it might sometimes be tempting to use these
highly crossreactive sera. This should preferably be avoided.

There are several reasons
why it is important that the content of potential allergens can be precisely
and sensitively determined in GMO foods. Since it is the ultimate goal to
secure safe food consumption for the individual patient, the allergic popula­tion,
and the society in total, both consumers, producers, health care personal, and
legislators should be able to have information on the concentration of
ingredients in a given food. It is important to emphasize, that this
information not only pertains to the actually products of modified genes, but
to all potential allergens, since the amount of gene products that are native
to the organism, may very well be changed by the introduction of a new or
changed gene in the organism.

Determination of
potential allergens by biological methods

The biological
activity of food allergens or mixtures thereof may be determined by various in
vivo and in vitro methods that may quantitatively or semi-quantitatively
express the combined effects of individual allergenic molecules in a mixture.
Even in the rare case of testing an individual food allergen molecule, a
response will emerge that is only declared relatively to other allergenic
substances or mixtures. Thus, an important feature of testing the biological
activity of mixtures is the lack a response which can be directly linked to
individual molecular entities, and this put special emphasis on the definition
of both the test systems and the mixtures that are tested.

The biological
activity of a substance in relation to food allergy may be understood in
various ways. In the context of this review only food allergic diseases
believed to be mediated by immunoglobulin E (IgE) will be considered even
though adverse reactions to foods include other disease entities and even the
term food allergy may comprise diseases elicited by several other mechanisms (1).

The term allergenic
may be understood both as the capacity to sensitize, i.e. induce an IgE immune
response, and as the capacity to elicit an allergic reaction in an individual
already sensitized. In this chapter only the latter meaning will be discussed
corresponding to the left branches of the decision trees. The induction of an
allergic reaction in an already sensitized individual, has been much more
successfully investigated and as described below numerous models exist for determination of the biological potency
(Table 1), which lists the various test procedures in the opposite direction of
what is seen in the decision trees .

Table 1.

TBL
1

Target

Species

Test
system

Examples

In
vivo

Entire
orga­nism

Human

Challenge
of allergic patients

DBPCFC,
open challenges

Experimental
animals

Peroral
challenge of animals

Anaphylactic
response

Skin

Human

Skin
testing of allergic pati­ents

Skin
prick tests, intrademal tests

Experimental
animals

Actively
or passively sensiti­zed animals

PCA

In
vitro

Ba­sophils

Human

Actively
or pas­sively sensiti­zed basophils + allergen

Basophil
hista­mine release, cord blood basophil histamine release

Basophil
or mast cells

Humanized,
i.e. trans-fected with a human IgE receptor

Passively
sensitized cells

Mediator
release

Mast
cells

Rodents

Peritoneal
mast cells

Histamine
or other mediator release

Modified
from In vivo and in vitro techniques to
determine the biological activity of food allergens (Review);Poulsen LK Submitted.

The Table illustrates
the hierarchy that exist among these test systems as challenge of human
patients are considered as closest to the relevant biological response, i.e.
elicitation of an actual allergic response, albeit under controlled and safe
circumstan­ces. The next level in the hierarchy is to use the skin as a
restricted and localized area for challenge. This system obviously involves the
skin mast cells, which must be sensitized by IgE in order to respond to the
offending allergen. Leaving the in vivo systems, the next step is to use the sensitized
basophil granulo­cyte as a model for the sensitized mast cell present in the
relevant organ of the patient. Moving further away from the actual patient,
basophil from a non-allergic donor such as cord blood may even be used as an
reagent which are then sensitized by IgE derived from an actual patient.

The above-mentioned
human model systems all have their animal experimental counterparts which will
not be further discussed since many of the parameters of the human systems
discussed below, will also apply to animal models. The basic problem with
experimental animals is to actually make them allergic. Although several
immunization schemes - often parenteral - are available, which will readily
produce an IgE response, it is still not known whether the mere presence of IgE
specific to a food allergen gives a good prediction of allergenicity.

The ultimate determination
of the biological activity of a food allergen or a mixture of these is the
effect on a sensitized food allergic patient. The first report on
double-blinded placebo controlled food challenges (DBPCFC) is probably a study
of May, where asthmatic children were challenged with freeze-dried foods in
capsules (2). By 1991 Bock and Atkins reviewed
about 500 challenges performed at their pediatric centre, and a pattern emerged
with relatively few placebo reactions and a high degree of safety (3). Thus DBPCFC has been said to be the gold standard
of food hypersensitivity diagnosis (4), and is
recommended by the European Academy for Allergy and Clinical Immunology as the only conclusive evidence of a food
allergy, provided it is performed properly(1).

For both practical and
ethical reasons it is obvious, that patients cannot be routinely challenged to
potential allergenic preparations. Thus the main reason for conducting
controlled food challenges in patients is to verify or rule out a suspicion of
food allergy, establishing a clinical diagnosis for the benefit of the patient.
It is possible however to perform clinical trials on allergic patients in order
to obtain knowledge about the allergenicity of GMO foods, provided that all
other toxicologal and safety issues have been satisfactory solved..

Skin tests in humans

For the
standardization of inhalation allergens the skin test has been the most
important tool, and it is the recommended method for biological standardization
of allergen extracts (5; 6). For this reason skin
tests have also been used extensively for diagnosis of food allergy (1), and it seems well justified to use it for
biological activity measurements of food allergens including GMO-foods. The
rationale behind skin testing is that by introducing a small volume of allergen
in the skin - either intradermally or via a small puncture of the stratum corneum as in the skin prick
test - mast cells sensitized with specific IgE are activated via allergen
cross-linking of this IgE. The activation of mast cells results in release of
mediators - primarily histamine - which induces a wheal and flare reaction of
the skin. Within a certain concentration range there is a dynamic response,
i.e. a wheal and flare with a larger area develop after application of a higher
concentration of allergen. The biological response is measured by planimetry as
the area of the wheal or the flare (7; 8) and the
result may be quantified by end-point-titration, i.e. the highest concentration
in a titration which produces a negative response, or by comparison with a
standard, typically histamine in a concentration of 10 mg/ml. The patient must
- besides being well-defined as a patient - fulfill certain conditions such as
an intact skin, lack of dermo­graphism, and abstinence from drugs such as
antihistamines which will dramatically inhibit the skin reaction (9). For ethical and safety reasons the test substance
must be assured to be without infectious or toxicological potential besides its
allergenic properties. A detailed outline of the technique is given in the
guidelines, and will not be discussed here, but some points of special
relevance for food allergens and GMO foods should be mentioned, however. The
guidelines recommend the use of 20 patients with symptoms of moderate severity,
but with the number of available patients that have undergone a
DBPCFC-procedure this may present a problem in many centres, especially if -
for ethical reasons - only adults or adolescents are selected. Moreover,
infants and small children have a good prognosis for outgrowing their food
allergy (10) early in their life, which makes
them less suited for participation in safety studies. Due to the paucity of
DBPCFC+ patients and the individual respon­ses which may be quite varying, it
can be difficult to perform a sufficient number of skin tests to ensure safety
of the GMO.

As an alternative to the skin test the basophil granulocytes which are believed to be sensitized
analogously to skin mast cells have been used extensively for many
immunological studies of the allergic response (11,12). Being an in
vitro method this technique has obvious advantages compared to the skin
tests, since less strict requirements are posed on the test substance regarding
non-toxicity and non-infectivity, albeit it should not be cytotoxic
in the applied concentrations. For studies of inhalation and food allergy histamine
release tests are correlating well with other measures ofIgE sensitization, such as skin prick tests
or determination of specific IgE in plasma (13-16),
and based on these findings it has been suggested to use the technique for
biological standardization of allergen extracts including food allergens.

The direct histamine
release method uses blood from a sensitized patient, and this limits the
practical possibilities for running the method to patient-near centres, since
the whole blood must be used within 24 h after drawing of the blood. This
obstacle can be overcome by combining basophils from a non-sensitized person
with serum containing specific and relevant IgE-antibodies. In its original
form, basophils from adult donors were stripped of their original IgE by a
brief treatment with low pH, followed by a new incubation with the sensitizing
serum (12; 17). More recently cord blood
basophils have been used as recipient cells, which has the advantage of
eliminating the low pH IgE dissociation step, which may interfere with the
biological functions of the cells. It is conceivable that basophil cell lines,
such as the KU812 (18) or animal cell lines
transfected with the the human FcεRI, i.e. the IgE high affinity receptor (19), may also be used as recipient cells for this
purpose.

Determination of
potential allergens by biochemical methods

A number of systems
for biochemical detection of allergens are listed in Table 2. A pure system
related to the allergic patient can be obtained by immunochemical assays
detecting IgE-allergen binding directly or indirectly by inhibition designs.
More indirect methods involve the use of animal antibodies for immunochemical
detection, or molecular biology methods for detection via the DNA or mRNA
levels.

Modified
from In vivo and in vitro techniques to
determine the biological activity of food allergens (Review);Poulsen LK Submitted.

In vitro studies of
IgE-allergen binding: RAST and RAST-inhibition

Since the binding
between the allergen and the IgE is central in eliciting of the biological
function in the test systems described above, it is obvious to use a test
system that measures this binding, and the RadioAllergoSorbentTest and modifica­tions
of this play an important role in allergen standardization. The initial design
of the RAST was based on the use of dextran-derived materials (20; 21) but later solid phases have comprised the widely
used paper discs (22), aluminium hydroxide gel (23), polystyrene tubes (24),
cellulose polymers (25; 26), and magnetic
micropar­ticles (27). Reviews of the available
technologies and a discussion of method evaluation have been given in (28; 29).

Other immunochemical
assays

In the Table 2 is also
mentioned the production of animal antibodies to individual allergens and the
use of such antibodies in ELISAs etc, forms the border between the biological
assays and molecular identification of individual allergens A large number of
animal antibodies has been raised against known or suspected food allergens,
and may be used for testing. Several commercial assays for well-known food
allergens have been described (30-33) [More
references to be added, please give me all your inputs!] (summarized in Table
3) and more are to follow. A word of caution should be issued: If gene products
are only slightly modified, it is important to carefully check how an antibody
raised to the native protein (allergen) will react to the modified protein. In
some cases it may be necessary to raise new antibodies to the modified protein.

Molecular biology
assays

The final line in the
Table 2 mentions the possibility of using determination of mRNA or DNA as a
surrogate marker of the presence of allergens. Since the DNA may be transcribed
with varying efficiency in the plant and the correlates between mRNA levels and
protein levels may also vary, these measures may only be sei-qualitatively
related to the potential allergen level. On the other hand, the molecular
biology are very sensitive and may be the only way to determine extremely low
levels of (genes coding for) allergens in GMO foods. Moreover, since the
sequence of the targetted genes is often known, these techniques may be able to
differentiate between native and genetically modified versions of the same gene
products, since small dissimilari­ties may evade detection by immunochemically
based techniques.

10.Host, A., & Halken, S. (1990).A prospective study of cow milk allergy in
Danish infants during the first 3 years of life. Clinical course in relation to
clinical and immunological type of hypersensitivity reaction.Allergy,45, 587-596.

Adverse reactions to
foods may be toxic or non-toxic in nature (Bruijnzeel-Koomen 1995, Anderson
1996). Non-toxic adverse reactions are either non-immune-mediated (food
intolerance) or immune-mediated (food allergy). Allergic reactions may be
either IgE-mediated or non-IgE-mediated (Bousquet 1997). Only those proteins
which cause an IgE response in human can be assessed for the allergenicity in skin
testing and in in vitro IgE-binding tests.

Allergenic food
sources cover certain plant- and animal-derived foods. World-wide, 8 foods are
considered to be the most important food stuffs causing IgE-mediated allergy,
e.g. egg, milk, fish, crustacean, peanut, soybean, wheat, and tree nut (Sampson
1998). However, regional differences may exist depending on dietary references
and cross-reactivities with inhalant allergens. In addition to these most
common allergenic foods, many other foods are known to be allergenic such as
several fruits, vegetables, and spices.

Symptoms of the
IgE-mediated allergy typically involve the skin, respiratory and/or
gastrointestinal tract (Bruijnzeel-Koomen 1995, Sampson 1999). However, a
number of gastrointestinal food allergies are not IgE-mediated, but may be a
result of a lectin, irritant or other type of effect. Therefore, understanding
and identification of the mechanisms involved in adverse reactions to foods is
an important prerequisite in selecting patients for tests on allergenicity of a
genetically engineered food.

Often, the only
symptom from food allergy may be oral allergy syndrome (OAS) typically caused
by fresh fruits, vegetables and spices (Kazemi-Shirazi 1999). In about 30-40%
cases OAS is associated with pollen allergy (Bircher 1994). In OAS food
allergens are in direct contact with the oral mucosa.

Foods are the most
common cause of IgE-mediated anaphylaxis (Pumphrey 1996). World-wide, in fatal reactions
peanut is the most common causative food allergen (Sampson 1998). However, also
other foods such as milk, egg, fish, crustaceans, tree nuts, soy, celery, kiwi,
and wheat may cause anaphylaxis (Foucard 1999). In addition, the assessment of
the allergenicity of foods causing severe allergic reac­tions (anaphylaxis)
only in association with exercise (e.g. exersice-indused anaphy­laxis, EIA) may
turn out difficult (Shadick 1999).

The prevalence of food
allergy has been investigated in a limited number of studies. The prevalence of
food allergy among adult population is 1- 2 % (Sampson 1999). The figure is higher (up to 8%) among
children who, however, often outgrow their food allergy before the 3rd birthday
(Bock 1984). The prevalence figures are higher when OAS is taken into account.These phenomenena may hamper the
selection of appropriate subjects for in vivo testing of a novel food.

Another major problem
is the presence of cross-reactivity between various groups of foods, where
detection of specific IgE antibodies towards a cross reacting food may or may
not be reflectedcin the clinical situation. This create a need for evaluation
also of cross reacting potential of new foods.

Foods contain several
proteins, only a few of which are allergens. However, no fixed safety limit can
be assessed under which no allergic reactions would occur. It is thus important
to emphasize, that a safe lower level of allergenic foods (No Effect Level,
where even the most sensitive patient does not react) probably does not exist.

At the FAO/WHO
Consultations on Genetically modified foods in Rome, 1996, the following statements
were launched (ref in[1]):

1The
transfer of genes from com­monly allergenic foods should be discouraged unless
it can be documented that the gene transferred does not code for an allergen.

2Foods found
to contain an allergen transferred from the organism which provided the DNA
should not be considered for marketing approval unless such products can be
clearly identified in the marketplace and this identity will not be lost
through distribu­tion and processing. Fur­ther, that labelling approaches may
not be practical in these situations, and that particular problems exist for
consumers who cannot read, or who may not be provided with labels.

3Involved
organizations should consider the appropriateness of, and/or actions to take,
in respect to foods containing new protein(s) that are determined to have the
charac­teristics of an allergen.

4The
identification of food allergens and the characteristics of these allergens
that define their immunogenicity be encouraged.

The ILSI decision tree
so far constitute the only guideline for assessment of potential change in
allergenicity of genetically modified organisms[2]:

This decision tree
divides GMOs into foods where the sour­ce of gene stems from a known
allergenic sour­ce or a source not known to be allergenic. Furthermore, the
allergenic sour­ce is further subdivided into sources from the commonly
allergenic foods (the big eight) and less commonly allergenic foods, namely the
remaining foods, only accounting for approx. 10 per cent of the clinical reac­tions.
This latter statement is however, limited to the classical type I allergic
reactions, elicited by e.g. peanut, milk, egg, soy, fish, crusta­ceans but does
not take into account the much more abundant reactions to to cross- or common
reactivity between e.g. pollens and fruits/vegatab­les or Latex and fruits[3].

The left side of the
decision tree dealing with known allergenic proteins contains the test systems
sufficient to rule out a potential risk to the allergic patient, provided the
tests systems (both in vitro and in vivo) are used with material from high
quality patients, fulfilling the EAACI guidelines[4]
and using validated test proce­dures[5],[6].
The subdivision into common and less common foods is, however based on
availability of test material rather than an actual risk assessment and should
thus be left out - there are no data in literature supporting an increased risk
for the actual patient to common food allergens than to less common food
allergens.

­The right side of
the decision tree, fig 1, deals with inserted proteins, not known to be
allergenic:

This
aproach is based on the follo­wing assumptions:

1The optimal peptide length for bin­ding
appears to be between 8 and 12 amino acids for T-cell epitopes and even longer
for B-cell epito­pes[7]

2All epitopes are sequential, and
conformational epitopes are wit­hout significance

3All relevant epitopes has already been
sequenced.

4The stability to di­gestion is a
significant and valid parameter that distinguishes food aller­gens from
non-allergens[8].

None of these
statements has been proven- there are
examples of exceptions for all the above statements. Further­more, the test is
also likely to identify conserved sequences that are unrelated to the
allergenic potential of the proteins.Furthermore, harmless proteins might also
be excluded form market based on these tests.

The EAACI risk evaluation procedure

It therefore suggested
to add a screening procedure to be applied to gene modified foods, not
previously known to be allergenic (the right side), fig 2:

The various subsequent
steps in the evaluation procedure is commented in the following (The numbers
refer to the flow chart).

-The left side of the decision tree dealing
with known allergenic proteins contains the test systems sufficient to rule out
a potential risk to the allergic patient, provided the tests systems (both in
vitro and in vivo) are used with material from high quality patients,
fulfilling the EAACI guidelines and using validated test procedures.

After a negative outcome oftesting for sequence similarity to known aller­gens,
the food is subjected to solid phase immunoassays screning for allerge­nicity
using sera from patients with established allergy to major allergens,
especially allergens, where cross-reactivity to foods are abundant (pollen
allergics).

It is suggested to use
as a minimum 3 * 10 patients allergic to birch, grass, artemisia respectivelyor
to other relevant allergens.There are
major regional differences between reaction severity and plants in question
within Europe. Allergens shou­ld therefore be included according to origin e.g.
ragweed, Parietaria or other types of food or inhalant allergens according to
type of GM-food in qu­estion.

Positive results in
these tests trans­fer the evaluation of the food to the left side of the flow
chart.

Sero­logical
cross-reactions should also be dealt with by transferral to the left side of
the decision tree (see -).

This step constitute considerations on the more uncertain
aspects of a novel food. In this phase of evaluation, aspects like e.g. models
for evaluation of a possible immunogenetic role as weel as considerations on a
possible sensiti­zing potential­­, possiblestimulation of TH-2 system or
creation of Neo-allergens­­. Also, the anticipated dose of intake and other
aspects may be included.

Finally, the natural
variability of allergens in wild type foods must be taken into account when
assessing quantitative aspects of measurement of allergens in GMOs.

At present, the
methods for such evaluation procedures are not fully develo­ped. The upcome of
animal models, which at present are not sufficiently developed for use may
elucidate these aspects in the future.

After evaluation of the above parameters, it will be
possible for the authorities to perform a proper risk assessment of the GMO in
question.

The left side of the
flow chart contains various levels of safety. A positive outcome in step 
(DBPCFC) of course constitutes the highest possible risk, whereas previous
experience with safe ingestion diminishes the absolute risk.

The in vivo and in
vitro investigations (-) thus results in data concerning the absolute risk of
introduction of the GMO in question to the market, whereas data from  enables
a calculation on the relative risk at a certain level (deter­mined in-).